The present invention relates to a method and a reagent for quantitating D-cysteine.
Almost all amino acids existing in organisms have been considered to be L-amino acids until recently when a high amount of D-amino acids were found in higher animals including mammals. Since there is a growing interest in sources and roles of the D-amino acids in the fields of biotechnology, clinical test, medical science and the like, it has become very important to establish a method for quantitating D-amino acids in an easy, rapid and highly-sensitive manner.
D-cysteine is one kind of D-amino acids. Conventionally, D-cysteine in a sample was quantitated, for example, by high performance liquid chromatography.
The present invention aims at providing a method and a reagent for quantitating D-cysteine in a convenient and highly-sensitive manner.
We achieved the present invention by finding that D-cysteine can be quantitated by, first, reacting D-cysteine and a D-luciferin precursor, and then reacting D-luciferin produced by the first reaction with luciferase.
The present invention relates to a method for quantitating D-cysteine, and provides the following (i) to (v):
(i) A method for quantitating D-cysteine in a sample, comprising the steps of:
(a) reacting D-cysteine with a D-luciferin precursor to produce D-luciferin or a derivative thereof; and
(b) quantitating D-cysteine by determining the level of the produced D-luciferin or D-luciferin derivative.
(ii) A method for quantitating D-cysteine according to (i) above, wherein the step of determining the level of the produced D-luciferin or D-luciferin derivative is carried out by determining a level of luminescence generated upon reacting D-luciferin or a derivative thereof with a bioluminescent reagent.
(iii) A method for quantitating D-cysteine according to (i) above, wherein the D-luciferine precursor is 2-cyano-6-hydroxybenzothiazole or 2-cyano-6-O-xcex2-D-galactosylbenzothiazole.
(iv) A method for quantitating D-cysteine according to (iii) above, wherein the step of determining the level of the produced D-luciferin or D-luciferin derivative is carried out by determining a level of luminescence generated upon reacting D-luciferin or a derivative thereof with a bioluminescent reagent.
(v) A method for quantitating D-cysteine, comprising the steps of:
(a) reacting D-cysteine with 2-cyano-6-hydroxybenzothiazole to produce D-luciferin; and
(b) calculating a D-cysteine level by determining a level of luminescent generated upon reacting the produced D-luciferin with luciferase, ATP and magnesium ion.
The present invention also relates to a reagent for quantitating D-cysteine, and provides the following (vi) and (vii):
(vi) A reagent for quantitating D-cysteine, comprising a D-luciferin precursor.
(vii) A reagent for quantitating D-cysteine, comprising a D-luciferin precursor and a bioluminescent reagent.
This specification includes all or part of the contents as disclosed in the specification of Japanese Patent Application No. 11-79691, which is a priority document of the present application.